ES2710652T3 - New SMB process - Google Patents

Abstract

A chromatographic separation process to recover a polyunsaturated fatty acid (PUFA) product from a feed mixture, a process comprising the steps of: (i) purifying the feed mixture in a first separation stage in a chromatography apparatus of simulated or real moving bed having a plurality of joined chromatography columns containing, as eluent, an aqueous organic solvent, to obtain an intermediate product; and (ii) purifying the intermediate product obtained in (i) in a second separation stage using a simulated or real mobile bed chromatography apparatus having a plurality of joined chromatography columns containing, as eluent, an aqueous organic solvent, to obtain the PUFA product; wherein (a) the first and second separation stages are carried out sequentially in the same chromatography apparatus, the intermediate product being recovered between the first and second separation stages and the process conditions in the chromatography apparatus being adjusted between the first and second separation stages so that the PUFA product is separated from different components of the feed mixture at each separation stage; or (b) the first and second separation stages are carried out in a first and second independent chromatography apparatus respectively, the intermediate product obtained from the first separation stage being introduced into the second chromatography apparatus, and the PUFA product being separated of different components of the feed mixture at each stage of separation; and wherein the aqueous organic solvent eluent used in each separation step has a volume ratio of water: different organic solvent; and wherein (1) the intermediate product is collected as the refining stream in the first separation stage, and the PUFA product is collected as the extract stream in the second separation stage; or (2) the intermediate product is collected as the extract stream in the first separation stage, and the PUFA product is collected as the refining stream in the second separation stage.

Description

DESCRIPTION

New SMB process

The present invention relates to an improved chromatographic separation process for purifying polyunsaturated fatty acids (PUFA) and derivatives thereof. In particular, the present invention relates to an improved simulated or actual cell chromatographic separation process for purifying PUFAs and derivatives thereof. Fatty acids, in particular PUFAs, and their derivatives are precursors of biologically important molecules, which play an important role in the regulation of biological functions such as platelet aggregation, inflammation and immune responses. Therefore, PUFAs and their derivatives may be therapeutically useful in the treatment of a wide range of pathological conditions including CNS conditions; neuropathies, including diabetic neuropathy; cardiovascular diseases; general conditions of the immune system and inflammatory, including cutaneous inflammatory diseases.

PUFAs are found in natural raw materials, such as vegetable oils and marine oils. However, these PUFAs are frequently present in said oils mixed with saturated fatty acids and many other impurities. Therefore, in a desirable way, PUFAs must be purified before their nutritional or pharmaceutical uses.

Unfortunately, PUFAs are extremely fragile. Therefore, when heated in the presence of oxygen, they are prone to isomerization, peroxidation and oligomerization. Therefore, the fractionation and purification of PUFA products to prepare pure fatty acids is difficult. Distillation, even in vacuo, can lead to degradation in unacceptable products.

Simulated or actual movable cell chromatography are known techniques, familiar to those skilled in the art. The principle of operation involves the countercurrent movement of a liquid eluent phase and a solid adsorbent phase. This operation allows a minimum use of solvent making the process economically viable. Said separation technology has several applications in different areas, including hydrocarbons, industrial chemical compounds, oils, sugars and APIs.

As is well known, in a conventional stationary bed chromatographic system, a mixture whose components must be separated is percolated through a container. The container is usually cylindrical, and is generally called a column. The column contains a filling of a porous material (generally referred to as the stationary phase) which shows high fluid permeability. The rate of percolation of each component of the mixture depends on the physical properties of that component, so that the components leave the column successively and selectively. Therefore, some of the components tend to bind strongly to the stationary phase and, therefore, will percolate slowly, while others tend to settle weakly and leave the column more rapidly. Many different stationary bed chromatographic systems have been proposed and are used for both analytical and industrial production purposes.

In contrast, a simulated movable bed chromatographic system consists of a series of individual columns containing adsorbent which are connected together in series. The eluent is passed through the columns in a first direction. The injection points of the feed and the eluent, and the collection points of the separated components in the system, are periodically moved by means of a series of valves. The overall effect is to simulate the operation of a single column containing a mobile bed of solid adsorbent, the solid adsorbent moving in a countercurrent direction of the eluent flow. Therefore, a simulated mobile bed system consists of columns which, as in a conventional stationary bed system, contain stationary beds of solid adsorbent through which the eluent is passed, but in a simulated mobile bed system the operation is such that it simulates a continuous moving bed upstream. "

Processes and equipment for simulated mobile-bed chromatography are described in several patents, such as US-2,985,589, US-3,696,107, US-3,706,812, US-3,761,533, FR-A-2103302, FR-A-2651148 and FR-A-2651149. The subject is also discussed in more detail in the document "Preparative and Production Scale Chromatography", edited by Ganetsos and Barker, Marcel Dekker Inc, New York, 1993.

A real mobile bed system is similar in operation to a simulated mobile bed system. However, instead of displacing the injection points of the feed mixture and the eluent, and the collection points of the separated component by means of a valve system, instead a series of adsorption units (ie columns) they move physically with respect to the feeding and extraction points. Again, the operation is such that it simulates a continuous moving bed upstream.

Processes and equipment for real cell-bed chromatography are described in several patents, such as US Pat. No. 6,979,402, US Pat. No. 5,069,883 and US Pat. No. 4,764,276.

WO 2007/075499 discloses a method for preparing compositions using argon chromatography, using an argentized cationic resin or an argentized alumina conditioned to separate compounds containing saturated or monounsaturated carbon chains from compounds having polyunsaturated carbon chains present in a starting composition. WO 2007/075499 also discloses a method for preparing an alumina-conditioned alumina adsorbent having a higher selectivity for compounds containing one or more polyunsaturated carbon chains.

The purification of PUFA products is particularly difficult. Thus, many feedstocks suitable for preparing PUFA products are extremely complex mixtures containing a large number of different components with very similar retention times in chromatography apparatuses. Therefore, it is very difficult to separate certain PUFAs from said feeding loads. However, a high degree of purity of PUFA products is required, particularly for pharmaceutical and nutraceutical applications. Historically, therefore, when high purity PUFA products are required, distillation has been used. There are, however, important disadvantages associated with the use of distillation as a separation technique for delicate PUFAs as described above.

Heretofore, no chromatographic technique has been made available to achieve high purity PUFA products, for example with a purity of greater than 95 or 97%, in particular from commercially available feedstocks such as oil fish.

A typical simulated mobile bed chromatography apparatus is illustrated with reference to Figure 1. The concept of a simulated or actual movable cell chromatographic separation process is explained by considering a vertical chromatographic column containing a stationary phase S divided into sections, more specifically in four overlapping subzones I, II, III and IV that go from the bottom to the top of the column. The eluent is introduced in the lower part in IE by means of a pump P. The mixture of components A and B that must be separated is introduced in IA B between sub-zone II and sub-area III. An extract containing mainly B is collected in SB between subzone I and subzone II, and a raffinate containing mainly A in SA is collected between subzone III and subzone IV.

In the case of a simulated mobile bed system, a simulated downward movement of the stationary phase S is caused by the movement of the introduction and collection points with respect to the solid phase. In the case of a real moving bed system, a simulated downward movement of the stationary phase S is caused by the movement of the various chromatographic columns with respect to the points of introduction and collection. In Figure 1, the eluent flows upward and mixture A B is injected between subzone II and subzone III. The components will move according to their chromatographic interactions with the stationary phase, for example adsorption on a porous medium. The B component that shows a stronger affinity for the stationary phase (the component that advances more slowly) will be dragged more slowly by the eluent and will follow with a delay. Component A that shows the weakest affinity for the stationary phase (the fastest advancing component) will be easily dragged by the eluent. If the correct set of parameters, especially the flow rate in each sub-area, are correctly estimated and controlled, component A showing the weakest affinity for the stationary phase will be collected between sub-area III and sub-area IV as a raffinate and component B which shows the strongest affinity for the stationary phase will be collected between subzone I and subzone II as an extract.

It will be appreciated, therefore, that the conventional simulated mobile bed system illustrated schematically in Figure 1 is limited to a binary fractionation.

Accordingly, there is a need for a simulated or real movable bed chromatographic separation process that can separate PUFA or its derivatives from components that progress both rapidly and slowly (ie, more polar and less polar impurities), to produce PUFA products from high purity from commercially available feedstocks such as fish oils. It is desirable, in addition, that the process include economic eluyentes that work under normalized conditions of pressure and temperature.

Summary of the invention

It has now been surprisingly discovered that a PUFA product can be efficiently purified from commercially available feedstocks such as fish oils by a simulated or actual movable bed apparatus using an aqueous organic solvent eluent. The present invention therefore provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, a process comprising the steps of:

(i) purifying the feed mixture in a first step of separation in a simulated or real movable bed chromatography apparatus having a plurality of bound chromatography columns containing, as eluent, an aqueous organic solvent, to obtain an intermediate product ; Y

(ii) purifying the intermediate product obtained in (i) in a second separation step using a simulated or actual movable bed chromatography apparatus having a plurality of bound chromatography columns containing, as eluent, an aqueous organic solvent, for obtain the PUFA product; in which (a) The first and second separation steps are carried out sequentially in the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions being adjusted in the chromatography apparatus between the first and second steps. separation steps so that the PUFA product is separated from different components of the feed mixture in each separation step; or

(b) the first and second separation steps are carried out in a first and second independent chromatography apparatus respectively, introducing the intermediate product obtained from the first separation step in the second chromatography apparatus, and separating the PUFA product from different components of the feed mixture in each stage of separation; Y

wherein the aqueous organic solvent eluent used in each separation step has a volume ratio of water: different organic solvent; Y

wherein (1) the intermediate product is collected as the refining stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation step; or

(2) the intermediate product is collected as the extract stream in the first separation step, and the PUFA product is collected as the refining stream in the second separation step. Also disclosed herein is a PUFA product which can be obtained by the process of the present invention.

The PUFA products produced by the process of the present invention are produced in high yield, and have high purity. In addition, the content of the characteristic impurities that normally occur after the distillation of PUFA is very low. As used herein, the term "isomeric impurities" is used to indicate those impurities normally produced during the distillation of natural oils containing PUFA. These include isomers, peroxidation products and PUFA oligomerization.

The present invention also provides a computer program for controlling a chromatographic apparatus as defined herein, computer program containing a code means which, when executed, commands the apparatus to carry out the process of the invention.

Description of the Figures

Figure 1 illustrates the basic principles of a simulated or real movable bed process for separating a binary mixture.

Figure 2 illustrates a first preferred embodiment of the invention that is suitable for separating EPA from components that progress more rapidly and more slowly (ie, more polar and less polar impurities). Figure 3 illustrates a second preferred embodiment of the invention that is suitable for separating DHA from components that progress more rapidly and more slowly (ie, more polar and less polar impurities). Figure 4 illustrates in more detail the first preferred embodiment of the invention that is suitable for separating EPA from components that progress more rapidly and more slowly (ie, more polar and less polar impurities).

Figure 5 illustrates in more detail the second preferred embodiment of the invention which is suitable for separating DHA from components that progress more rapidly and more slowly (ie more polar and less polar impurities).

Figure 6 illustrates in more detail an alternative method for the first preferred embodiment of the invention that is suitable for separating EPA from components that progress more rapidly and more slowly (ie, more polar and less polar impurities).

Figure 7 illustrates in more detail an alternative method for the second preferred embodiment of the invention that is suitable for separating DHA from components that progress more rapidly and more slowly (ie, more polar and less polar impurities).

Figure 8 illustrates a particularly preferred embodiment of the invention for purifying EPA of components that progress more rapidly and more slowly (ie, more polar and less polar impurities).

Figure 9 illustrates an alternative method for a particularly preferred embodiment of the invention for purifying EPA of components that progress more rapidly and more slowly (ie more polar and less polar impurities).

Figure 10 illustrates three ways in which the chromatographic separation process can be carried out. the invention.

Figure 11 shows an HPLC analysis of an EPA-rich feedstock that can be suitably used as the feed mixture in the process of the present invention.

Figure 12 shows an HPLC analysis of the raffinate (R) and extract (E) streams obtained in the first stage of separation of a process according to the present invention.

Figure 13 shows an HPLC analysis of the raffinate (R) and extract (E) streams obtained in the second stage of separation of a process according to the present invention.

Figure 14 shows a more detailed HPLC analysis of the extract stream obtained in the second stage of separation of a process according to the present invention.

Figure 15 shows a GC FAMES fingerprint of a DHA product produced by SMB.

Figure 16 shows a GC FAMES fingerprint of a DHA product produced by distillation.

Detailed description of the invention

The chromatographic separation process of the invention is usually different from a chromatographic separation process for the recovery of a polyunsaturated fatty acid (PUFA) product from a feed mixture, a process comprising introducing the feed mixture into a chromatography apparatus. of simulated or real movable bed having a plurality of bound chromatography columns containing, as eluent, an aqueous alcohol, wherein the apparatus has a plurality of zones comprising at least a first zone and a second zone, each zone having an extract stream and a refining stream from which liquid can be collected from said plurality of bound chromatography columns, and wherein (a) a refining stream containing the PUFA product together with more polar components is collected of a column in the first zone and is introduced into a non-adjacent column in the second zone, and / or (b) an extract stream that containing the PUFA product together with less polar components is collected from a column in the second zone and introduced into a non-adjacent column in the first zone, said PUFA product being separated from different components of the feed mixture in each zone.

As used herein in this embodiment, the term "zone" refers to a plurality of attached chromatography columns which contain, as eluent, an aqueous alcohol, and which have one or more injection points for a stream of feed mixture, one or more injection points for water and / or alcohol, a refining extraction stream from which liquid can be collected from said plurality of attached chromatography columns, and an extract stream from extract from from which liquid can be collected from said plurality of attached chromatography columns. Normally, each zone has only one injection point for a feed mix. In one embodiment, each zone has only one injection point for the aqueous alcohol eluent. In another embodiment, each zone has two or more injection points for water and / or alcohol.

Further details of this embodiment can be found in the international patent application No. PCT / GB 10/002339. The chromatographic separation process of the invention is normally different from the processes disclosed in PCT / GB 10/002339.

As used herein, the term "PUFA product" refers to a product that comprises one or more polyunsaturated fatty acids (PUFA), and / or derivatives thereof, usually of nutritional or pharmaceutical importance. Normally, the PUFA product is a unique PUFA or derivative thereof. Alternatively, the PUFA product is a mixture of two or more PUFAs or derivatives thereof, for example two. The term "polyunsaturated fatty acid" (PUFA) refers to fatty acids that contain more than one double bond. Said PUFAs are well known to the person skilled in the art. As used herein, a PUFA derivative is a PUFA in the form of a mono-, di- or triglyceride, ester, phospholipid, amide, lactone or salt. Triglycerides and esters are preferred. More esters are preferred. The esters are usually alkyl esters, preferably C ¹ -C ⁶ aliphatic esters, more preferably C ¹ -C ⁴ aliphatic esters. Examples of esters include methyl and ethyl esters. Ethical esters are the most preferred.

Typically, the PUFA product comprises at least one w-3 or w-6 PUFA, preferably at least one w-3 PUFA. Examples of PUFA or> -3 include alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid ( DHA). SDA, EPA and DHA ^d P are preferred. More EPA and DHA are preferred. Examples of PUFA w-6 include linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA), docosadienoic acid, adrenic acid and docosapentaenoic acid ( u> -6). LA, ARA, GLA and DGLA are preferred.

In one embodiment, the PUFA product is EPA and / or ethyl ester (EE) of the EPA.

In another embodiment, the PUFA product is DHA and / or ethylene ester (EE) of DHA.

In a further embodiment, the PUFA product is a mixture of EPA and DHA and / or EPA EPA and DHA EE. In a most preferred embodiment, the PUFA product is EPA or EPA ethyl ester which is produced with a purity greater than 90%, preferably with a purity greater than 95%, and more preferably with a purity greater than 97%.

Normally, in addition to said PUFA product, an additional secondary PUFA product is collected in the chromatographic separation process of the invention. Preferably, the PUFA product is EPA and the additional secondary PUFA product is DHA.

In a further embodiment of the invention, the apparatus is configured to collect a PUFA product which is a concentrated mixture of EPA and DHA. Therefore, a feed mix is used that contains EPA, DHA, components that are more polar than EPA and DHA, and components that are less polar than EPA and DHA. In the first separation stage, the less polar material than EPA and DHA is normally removed. In the second separation stage, the material that is more polar than EPA and DHA is normally removed, and a concentrated mixture of EPA and DHA is collected as the PUFA product.

Feeding mixtures suitable for fractionation by the process of the present invention can be obtained from natural sources including vegetable and animal oils and fats, and from synthetic sources including oils obtained from plants, animals and microorganisms including genetically modified yeasts. Examples include fish oils, algae oils and microalgae and vegetable oils, for example borage oil, Echium oil and evening primrose oil. In one embodiment, the feed mixture is a fish oil. In another embodiment, the feed mixture is an oil of algae. Seaweed oils are particularly suitable when the desired PUFA product is EPA and / or DHA. The genetically modified safflower oil is particularly suitable when the desired PUFA product is GLA. The genetically modified yeast is particularly suitable when the desired PUFA product is EPA.

In a particularly preferred embodiment, the feed mixture is a fish oil or feedstock derived from fish oil. It has been advantageously discovered that, when a fish oil or feed charge derived from fish oil is used, an EPA PUFA product or EPA ethyl ester can be produced by the process of the present invention with more than 90% purity. , preferably more than 95% pure, and more preferably more than 97% pure.

The feed mixture can be subjected to chemical treatment before fractionation by the process of the invention. For example, it can be subjected to transesterification with glycerides or glyceride hydrolysis followed in some cases by selective processes such as crystallization, molecular distillation, fractionation with urea, extraction with silver nitrate or other solutions of metal salts, iodolactonization or fractionation with supercritical fluid. Alternatively, a feed mix can be used directly without any initial treatment step.

The feed mixes usually contain the PUFA product and at least one more polar component and at least one less polar component. The less polar components have a stronger adhesion to the adsorbent used in the process of the present invention than that of the PUFA product. During operation, said less polar components normally move with the adsorbent phase solid in preference to the liquid eluant phase. The more polar components have a weaker adhesion to the adsorbent used in the process of the present invention than that of the PUFA product. During operation, said more polar components normally move with the liquid eluting phase in preference with respect to the solid adsorbent phase. In general, the more polar components will be separated in a refining stream, and the less polar components will be separated in an extract stream.

Examples of the more and less polar components include (1) other compounds that exist in natural oils (eg marine oils or vegetable oils), (2) by-products formed during the storage, refining and pre-concentration stages and (3) contaminants from solvents or reagents that are used during previous stages of concentration or purification.

The examples of (1) include other unwanted PUFAs; saturated fatty acids; sterols, for example cholesterol; vitamins; and environmental contaminants, such as polychlorinated biphenyls (PCB), polyaromatic hydrocarbon (PAH) pesticides, chlorinated pesticides, dioxins and heavy metals. PCB, PAH, dioxins and chlorinated pesticides are all highly non-polar components.

Examples of (2) include isomers and products of oxidation or decomposition of the PUFA product, for example, polymeric auto-oxidation products of fatty acids or their derivatives.

Examples of (3) include urea that can be added to remove saturated or monounsaturated fatty acids from the feed mixture.

Preferably, the feed mixture is a marine oil containing PUFA (for example a fish oil), more preferably a marine oil (for example a fish oil) comprising EPA and / or DHA. A typical feed mixture for preparing (EE) EPA concentrate by the process of the present invention comprises 50-75% of (EE) EPA, from 0 to 10% of (EE) DHA, and other components including other fatty acids or> -3 yu> -6 essentials.

A preferred feed mix for preparing (EE) EPA concentrate by the process of the present invention comprises 55% of (EE) EPA, 5% of (EE) DHA, and other components including other fatty acids u> -3 and w- 6 essentials. (EE) DHA is less polar than (EE) EPA.

A typical feed mixture for preparing the concentrated (EE) DHA by the process of the present invention comprises 50-75% (EE) DHA, from 0 to 10% (EE) EPA, and other components including other fatty acids or> -3 yu> -6 essentials.

A preferred feed mix for preparing (EE) DHA concentrate by the process of the present invention comprises 75% of (EE) DHA, 7% (EE) EPA and other components including other essential fatty acids u> -3 and w-6 . (EE) EPA is more polar than (EE) DHA.

A typical feed mixture for preparing a concentrated mixture of (EE) EPA and (EE) DHA by the process of the present invention comprises more than 33% of (EE) EPA, and more than 22% of (EE) DHA.

Each step of separation of the process of the present invention is carried out in a simulated or real movable bed chromatography apparatus.

Any known simulated or real movable bed chromatography apparatus may be used for the purposes of the method of the present invention, provided that the apparatus is used in accordance with the process of the present invention. Those apparatuses described in the patents US-2,985,589, US-3,696,107, US-3,706,812, US-3,761,533, FR-A-2103302, FR-A-2651148, FR-A-2651149, US- 6,979,402, 5,069,883 and 4,764,276 all may be used if they are configured in accordance with the process of the present invention.

As used herein, the term "simulated or real movable bed chromatography apparatus" usually refers to a plurality of attached chromatography columns containing, as eluent, an aqueous organic solvent, and having one or more injection points for a feed mix stream, one or more injection points for water and / or organic solvent, a refining extraction stream from which liquid can be collected from said plurality of attached chromatography columns, and a extract extraction stream from which liquid can be collected from said plurality of attached chromatography columns.

The chromatography apparatus used in each step of the process of the present invention has a unique set of chromatograph columns linked in series containing, as eluent, an aqueous organic solvent. Normally, each of the chromatography columns is attached to the two columns in the apparatus adjacent to that column. Therefore, the output of a given column of the set is connected to the input of the adjacent column of the group, which is downstream with respect to the eluent flow of the group. Therefore, the eluent can flow around the group of attached chromatography columns. Normally, none of the chromatography columns are attached to non-adjacent columns in the apparatus.

As used herein the term "non-adjacent" refers to columns, in for example the same apparatus, separated by one or more columns, preferably 3 or more columns, more preferably 5 or more columns, most preferably approximately 5 columns

Normally, each device has only one injection point for a feed mix. In one embodiment, each apparatus has only one injection point for the aqueous organic solvent eluent. In another embodiment, each apparatus has two or more injection points for water and / or organic solvent.

The term "refined" is well known to the person skilled in the art. In the context of real and simulated cell-bed chromatography, it refers to the stream of components that move more rapidly with the liquid eluent phase compared to the solid adsorbent phase. Thus, a refining stream is usually enriched with more polar components, and impoverished in less polar components as compared to a feed stream.

The term "extract" is well known to the person skilled in the art. In the context of real and simulated cell-bed chromatography, it refers to the flow of components that move more rapidly with the adsorbent phase solid in comparison with the liquid eluent phase. Therefore, an extract stream is normally enriched in less polar components, and impoverished in more polar components as compared to a feed stream.

The number of columns used in each apparatus is not particularly limited. A person skilled in the art would be able to easily determine an appropriate number of columns to use. The number of columns is normally 4 or more, preferably 6 or more, more preferably 8 or more, for example 4, 5, 6, 7, 8, 9 or 10 columns. In a preferred embodiment, 5 or 6 columns are used, more preferably 6 columns. In another preferred embodiment, 7 or 8 columns are used, more preferably 8 columns. Normally, there are no more than 25 columns, preferably no more than 20, most preferably no more than 15.

The chromatographic apparatuses used in the first and second separation stages usually contain the same number of columns. For some applications, there may be different numbers of columns.

The dimensions of the columns used in the apparatus are not particularly limited, and will depend on the volume of feed mixture to be purified. A person skilled in the art would be able to easily determine columns of an appropriate size to be used. The diameter of each column is usually between 10 and 1000 mm, preferably between 10 and 500 mm, more preferably between 25 and 250 mm, even more preferably between 50 and 100 mm, and most preferably between 70 and 80 mm. The length of each column is usually between 10 and 300 cm, preferably between 10 and 200 cm, more preferably between 25 and 150 cm, even more preferably between 70 and 110 cm, and most preferably between 80 and 100 cm.

The columns in the chromatographic apparatus used in the first and second separation stages usually have identical dimensions but may have, for some applications, different dimensions.

The flows introduced into the column are limited by maximum pressures through the series of columns and will depend on the dimensions of the column and the particle size of the solid phases. One skilled in the art will be able to easily establish the flow rate required for each column dimension to ensure efficient desorption. Columns of larger diameter will generally need higher flows to maintain linear flow through the columns.

For the typical column sizes described above, normally the flow rate of the eluent that is introduced into the chromatographic apparatus used in the first separation step is from 1 to 4.5 l / min, preferably from 1.5 to 2.5. l / min Normally, the flow rate of the extract from the chromatographic apparatus used in the first separation stage is from 0.1 to 2.5 l / min, preferably from 0.5 to 2.25 l / min. In embodiments where part of the extract from the first separation stage is recirculated back into the interior of the apparatus used in the first separation stage, the recirculation flow rate is usually from 0.7 to 1.4 l / min, preferably about 1. l / min Normally, the flow rate of the raffinate from the chromatographic apparatus used in the first separation step is from 0.2 to 2.5 l / min, preferably from 0.3 to 2.0 l / min. In embodiments where part of the refining from the first separation stage is recirculated back into the interior of the apparatus used in the first separation stage, the recirculation flow rate is normally from 0.3 to 1.0 l / min, preferably approximately 0 , 5 l / min. Typically, the rate of introduction of the feed mixture into the chromatographic apparatus used in the first separation step is from 5 to 150 ml / min, preferably from 10 to 100 ml / min, more preferably from 20 to 60 ml / min.

For the typical column sizes described above, normally the flow rate of the eluent introduced into the chromatographic apparatus used in the second separation step is from 1 to 4 l / min, preferably from 1.5 to 3.5 l / min. Normally, the flow rate of the extract from the chromatographic apparatus used in the second separation stage is from 0.5 to 2 l / min, preferably from 0.7 to 1.9 l / min. In embodiments where part of the extract from the second separation stage is recirculated back into the interior of the apparatus used in the second separation stage, the recirculation flow rate is usually from 0.6 to 1.4 l / min, preferably from 0 , 7 to 1.1 l / min, more preferably about 0.9 l / min. Normally, the flow rate of the raffinate from the chromatographic apparatus used in the second separation step is from 0.5 to 2.5 l / min, preferably from 0.7 to 1.8 l / min, more preferably approximately 1.4 l / min. / min. In embodiments where part of the refining from the second separation stage is recirculated back into the interior of the apparatus used in the second separation stage, the recirculation flow rate is usually from 0.3 to 1.0 l / min, preferably approximately 0 , 5 l / min.

As the person skilled in the art will appreciate, references to the flows to which liquid is collected or removed by the various streams of extract and refining refer to volumes of liquid removed in a quantity of time, usually 1 / minute. Analogously, references to the flow rates to which liquid is recirculated back into an apparatus, usually to an adjacent column in the apparatus, refer to liquid volumes recirculated in a quantity of time, usually l / minute.

The stage time, ie the time between the displacement of the injection points of the feed and eluent mixture, and the various extraction points of the fractions collected, is not particularly limited, and will depend on the number and dimensions of the used columns, and the flow that flows through the apparatus. A person skilled in the art will be able to easily determine the appropriate stage times for use in the process of the present invention. The stage time is usually from 100 to 1000 seconds, preferably from 200 to 800 seconds, more preferably from about 250 to about 750 seconds. In some embodiments, an appropriate stage time is from 100 to 400 seconds, preferably from 200 to 300 seconds, more preferably approximately 250 seconds. In other embodiments, an appropriate stage time is 600 to 900 seconds, preferably 700 to 800 seconds, more preferably approximately 750 seconds.

In the process of the present invention, real cell-bed chromatography is preferred.

In the process of the present invention, conventional adsorbents known in the art can be used for real and simulated mobile bed systems. Each chromatographic column may contain the same or a different adsorbent. Normally, each column contains the same adsorbent. Examples of such commonly used materials are polymeric beads, preferably polystyrene cross-linked with DVB (divinylbenzene); and silica gel, preferably reversed phase silica gel bonded to C8 or C18 alkanes, especially C18. The reverse phase silica gel bonded to C18 is preferred. The adsorbent using in the process of the present invention is preferably non-polar.

The shape of the adsorbent stationary phase material can be, for example, spherical or non-spherical beads, preferably substantially spherical beads. Said beads usually have a diameter of 5 to 500 micrometers, preferably of 10 to 500 micrometers, more preferably of 15 to 500 micrometers, more preferably of 40 to 500 micrometers, more preferably of 100 to 500 micrometers, more preferably of 250 to 500 micrometers , even more preferably from 250 to 400 micrometers, most preferably from 250 to 350 micrometers. In some embodiments, beads with a diameter of 5 to 35 micrometers, usually 10 to 30 micrometers, preferably 15 to 25 micrometers, may be used. Some preferred particle sizes are somewhat larger than the particle sizes of beads used in the past in simulated and real movable bed processes. The use of larger particles allows a lower eluent pressure to be used in the system. This, in turn, has advantages in terms of cost savings, efficiency and useful life of the device. It has been surprisingly discovered that large particle size adsorbent beads can be used in the process of the present invention (with its associated advantages) without any loss of resolution.

The adsorbent typically has a pore size of 10 to 50 nm, preferably 15 to 45 nm, more preferably 20 to 40 nm, most preferably 25 to 35 nm.

Typically, the process of the present invention is carried out at a temperature of 15 to 55 ° C, preferably 20 to 40 ° C, more preferably at about 30 ° C. Therefore, the process is carried out normally at room temperature, but can be carried out at elevated temperatures.

The process of the present invention comprises a first and a second separation stage.

These two steps can be carried out easily in a single chromatographic apparatus. Therefore, in one embodiment, (a) the first and second separation steps are carried out sequentially in the same chromatography apparatus, the intermediate product being recovered between the first and second separation stages and the process conditions being adjusted in the chromatography apparatus between the first and second separation stages so that the PUFA product is separated from different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10a. Therefore, the first separation stage (left side) is carried out in an SMB apparatus having 8 columns. Between the first and second separation steps the intermediate product is recovered in, for example, a vessel, the process conditions being adjusted in the chromatography apparatus so that the PUFA product is separated from different components of the feed mixture in each case. separation stage. The second separation stage (right side) is then carried out in the same SMB device that has 8 columns.

In embodiment (a), adjusting the process conditions usually refers to adjusting the process conditions in the apparatus as a whole, i.e. physically modifying the apparatus so that the conditions are different. It does not refer to simply reintroducing the intermediate product back into a different part of the same apparatus where the process conditions might be different.

Alternatively, a first and a second independent chromatography apparatus can be used in the first and second separation stages. Therefore, in another embodiment, (b) the first and second separation steps are carried out in a first and a second independent chromatography apparatus respectively, the intermediate product being obtained from the first separation step introduced in the second apparatus. of chromatography, and the PUFA product being separated from different components of the feed mixture in each separation step.

In embodiment (b), the two separation steps can be carried out sequentially or simultaneously.

Therefore, in embodiment (b) in the case where the two separation steps are carried out sequentially, the first and second separation steps are carried out sequentially in a first and a second independent chromatography apparatus respectively, recovering the intermediate product between the first and second separation stages and adjusting the process conditions in the first and second chromatography apparatuses so that the PUFA product is separated from different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10b. Therefore, the first separation stage (left side) is carried out in an SMB device having 8 columns, from one to eight. Between the first and second separation stages, the intermediate product is recovered, for example in a container, and then introduced into a second independent SMB apparatus. The second separation stage (right side) is carried out in the second independent SMB device that has 8 columns, from nine to sixteen. The process conditions in the two chromatography apparatuses are adjusted so that the PUFA product is separated from different components of the feed mixture in each separation step.

In the embodiment (b) in the case where the two separation steps are carried out simultaneously, the first and second separation steps are carried out in a first and a second independent chromatography apparatus respectively, the intermediate product being introduced in the chromatography apparatus used in the second separation step, and the process conditions being adjusted in the first and second chromatography apparatuses so that the PUFA product is separated from different components of the feed mixture in each separation step. A preferred embodiment of this separation process is shown as Figure 10c. Therefore, the first separation stage (left side) is carried out in an SMB device having 8 columns, from one to eight. The intermediate product obtained in the first separation step is then introduced into the second independent chromatography apparatus used in the second separation step. The intermediate product can be passed from the first separation stage to the second separation stage directly or indirectly, for example by means of a container. The second separation stage (right side) is carried out in the second independent SMB device that has 8 columns, from nine to sixteen. The process conditions in the two chromatography apparatuses are adjusted so that the PUFA product is separated from different components of the feed mixture in each separation step.

In embodiment (b) in the case where the two separation steps are carried out simultaneously, the eluent circulates separately in the two independent chromatographic apparatuses. Therefore, the eluent is not shared between the two independent chromatographic apparatuses apart from the fact that the eluent may be present as a solvent in the intermediate product which is purified in the second separation step, and which is introduced into the chromatographic apparatus used in the Second stage of separation. The chromatographic columns are not shared between the two independent chromatographic apparatuses used in the first and second separation stages.

After obtaining the intermediate product in the first separation step, the aqueous organic solvent eluent can be partially or completely removed before the intermediate product is purified in the second separation step. Alternatively, the intermediate product can be purified in the second separation step without the removal of any solvent present.

As mentioned above, the PUFA product is separated from different components of the feed mixture in each separation step. In embodiment (a), the process conditions of the individual SMB apparatus used in both separation stages are adjusted between the first and second separation stages so that the PUFA product is separated from different components of the feed mix at each stage of separation. In embodiment (b), the process conditions in the two independent chromatography apparatuses used in the first and second separation stages are adjusted so that the PUFA product is separated from different components of the feed mixture at each stage of separation.

Therefore, the process conditions in the first and second stages of separation vary. Process conditions that vary may include, for example, the size of the columns used, the number of columns used, the fill used in the columns, the stage time of the SMB apparatus, the temperature of the apparatus, or the eluent used. in the separation stages.

The intermediate product obtained in the first stage of separation is normally enriched in the PUFA product compared to the feed mixture.

The intermediate product obtained in the first separation stage is then introduced into the chromatographic apparatus used in the second separation stage.

The intermediate product is normally collected as the refining stream or extract from the chromatographic apparatus used in the first separation process.

In one embodiment, (1), the intermediate product is collected as the refining stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation step. Therefore, the refining stream collected in the first separation stage is used as a feed mix in The second stage of separation. The refining stream collected in the first separation stage usually contains the PUFA product together with more polar components.

In another embodiment, (2), the intermediate product is collected as extract stream in the first separation step, and the PUFA product is collected as the raffinate stream in the second separation step. Therefore, the extract stream collected in the first separation stage is used as the feed mixture in the second separation stage. The extract stream collected in the first separation step usually contains the PUFA product together with less polar components.

The PUFA product is separated from different components of the feed mixture at each stage of separation. Normally, the separate components in each step of separation of the process of the present invention have different polarities.

Preferably, the PUFA product is separated from less polar components of the feed mixture in the first separation step, and the PUFA product is separated from more polar components of the feed mixture in the second separation step.

Normally, (a) part of the extract stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation stage; and / or (b) part of the refining stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation stage; and / or (c) part of the extract stream from the apparatus used in the second separation step is recirculated back into the interior of the apparatus used in the second separation stage; and / or (d) part of the raffinate stream from the apparatus used in the second stage of separation is recirculated back into the interior of the apparatus used in the second stage of separation.

Preferably, (a) part of the extract stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation stage; and (b) part of the refining stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation step; and (c) part of the extract stream from the apparatus used in the second separation step is recirculated back into the interior of the apparatus used in the second separation stage; and (d) part of the raffinate stream from the apparatus used in the second stage of separation is recirculated back into the interior of the apparatus used in the second stage of separation.

This recirculation involves feeding part of the extract or refined stream out of the chromatography apparatus used in the first or second separation stage back into the apparatus used in that step, usually in an adjacent column. This adjacent column is the adjacent column that is downstream with respect to the eluent flow in the system.

The flow rate to which the liquid collected by the extract or refined stream in the first or second separation stages is recirculated back into the chromatography apparatus used in that step is the flow rate at which the liquid collected by that stream is fed from turn inside the apparatus used in that stage, usually inside an adjacent column, ie the column that is downstream with respect to the flow of eluent in the system.

This can be seen with reference to a preferred embodiment in Figure 9. The flow rate of extract recirculation in the first stage of separation is the flow rate at which the extract collected from the bottom of column 2 of the chromatographic apparatus used in the first stage of separation is fed into the top of column 3 of the chromatographic apparatus used in the first separation stage, ie the liquid flow rate into the upper part of column 3 of the chromatographic apparatus used in the first separation stage.

The flow rate of extract recirculation in the second stage of separation is the flow rate with which the extract collected in the lower part of column 2 of the chromatographic apparatus used in the second stage of separation is fed into the upper part of the column 3 of the chromatographic apparatus used in the second separation stage, ie the flow rate of liquid introduced into the interior of the upper part of column 3 of the chromatographic apparatus used in the second separation stage.

The recirculation of the extract and / or refined streams in the first and / or second separation stages is normally carried out by feeding the liquid collected by that stream in that separation stage into a vessel, and then pumping a quantity of that liquid from the container back into the interior of the apparatus used in that stage of separation, typically into an adjacent column. In this case, the flow rate of liquid recirculation collected by a particular extract or refining stream in the first and / or second separation stages, normally back inside an adjacent column, is the flow rate at which the liquid is pumped out. of the container back into the interior of the chromatography apparatus, typically into an adjacent column.

As the person skilled in the art will appreciate, the amount of liquid that is introduced into a chromatography apparatus by the eluent streams and feed charge is balanced with the amount of liquid removed from the apparatus, and recirculated back into the interior of the apparatus.

Therefore, with reference to Figure 9, for the extract stream, the flow rate of eluent (desorbent) introduced into the interior of the chromatographic apparatus or apparatus used in the first and second separation stages (D) is equal to the flow rate at which liquid collected by the extract stream in that separation step is accumulated in a vessel (E1 and E2) added to the stream at which the extract is recirculated back into the chromatographic apparatus used in that particular separation stage (D- E1 and D-E2).

For the refining stream from a separation step, the flow rate at which the extract is recirculated back into the chromatographic apparatus used in that particular separation stage (D-E1 and D-E2) added to the flow rate at which it is introduced the feed charge in the chromatographic apparatus used in that particular separation stage (F and R1) is equal to the flow rate at which the liquid collected by the refining stream in that particular separation stage accumulates in a vessel (R1 and R2) added to the flow rate at which the raffinate is recirculated back into the chromatographic apparatus used in that particular separation step (D + F-E1-R1 and D + R1-E2-R2).

The flow rate at which the liquid collected from a particular extract or refining stream from a chromatography apparatus is accumulated in a container can also be considered as the net rate of removal of that stream of extract or refined from that chromatography apparatus.

The eluent used in the process of the present invention is an aqueous organic solvent.

The aqueous organic solvent usually comprises water and one or more alcohols, ethers, esters, ketones or nitriles, or mixtures thereof.

The alcohol solvents are well known to the person skilled in the art. The alcohols are usually short chain alcohols. The alcohols are usually of the ROH formula, wherein R is a linear or branched C ¹ -C ⁶ alkyl group. The C ¹ -C ⁶ alkyl group is preferably unsubstituted. Examples of alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol. Preferred are methanol and ethanol. Methanol is more preferred.

Ether solvents are well known to the person skilled in the art. The ethers are usually short chain ethers. The ethers are usually of the formula RO-R ', in which R and R' are the same or different and represent a linear or branched C ¹ -C ⁶ alkyl group. The C ¹ -C ⁶ alkyl group is preferably unsubstituted. Preferred ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether (MTBE).

The ester solvents are well known to the person skilled in the art. The esters are normally short chain esters. The esters are usually of formula R- (C = O) O-R ', wherein R and R' are the same or different and represent a linear or branched C ¹ -C ⁶ alkyl group. Preferred esters include methyl acetate and ethyl acetate.

Ketone solvents are well known to those skilled in the art. Ketones are usually short chain ketones. Ketones are normally of formula R- (C = O) -R ', wherein R and R' are the same or different and represent a linear or branched C ¹ -C ⁶ alkyl group. The C ¹ -C ⁶ alkyl group is preferably unsubstituted. Preferred ketones include acetone, methyl ethyl ketone and methyl isobutyl ketone (MIBK).

The nitrile solvents are well known to the person skilled in the art. Nitriles are usually short-chain nitriles. The nitriles are normally of formula R-CN, where R represents a linear or branched C ¹ -C ⁶ alkyl group. The C ¹ -C ⁶ alkyl group is preferably unsubstituted. Preferred nitriles include acetonitrile. Typically, the aqueous organic solvent is aqueous alcohol or aqueous acetonitrile.

The aqueous organic solvent is preferably aqueous methanol or aqueous acetonitrile. Aqueous methanol is more preferred.

Normally, the eluent is not in a supercritical state. Normally, the eluent is a liquid.

Normally, the average ratio of water: organic solvent, eg ratio of water: methanol, of the eluent throughout the apparatus is from 0.1: 99.9 to 9:91 parts by volume, preferably 0.25: 99, 75 to 7:93 parts by volume, more preferably from 0.5: 99.5 to 6:94 parts by volume.

When the aqueous organic solvent is aqueous acetonitrile, the eluent normally contains up to 30% by weight of water, the rest acetonitrile. Preferably, the eluent contains from 5 to 25% by weight of water, the rest acetonitrile. More preferably, the eluent contains from 10 to 20% by weight of water, the rest acetonitrile. Even more preferably, the eluent contains from 15 to 25% by weight of water, the rest acetonitrile.

The eluent of aqueous organic solvent used in each separation step has a water: organic solvent ratio. The ratio of water: organic solvent used in each stage of separation is preferably adjusted so that the PUFA product can be separated from different components of the feed mixture in each stage of separation.

The elution power of the eluent used in each of the separation steps is usually different. Preferably, the elution power of the eluent used in the first separation step is greater than that of the eluent used in the second separation step. In practice, this is achieved by varying the relative amounts of water and organic solvent used in each stage of separation.

Depending on the choice of organic solvent, they can be more powerful desorbentes than water. As an alternative, they can be desorbent less potent than water. Acetonitrile and alcohols, for example, are more powerful desorbers than water. Therefore, when the aqueous organic solvent is an aqueous alcohol or acetonitrile, the amount of alcohol or acetonitrile in the eluent used in the first stage of separation is usually greater than the amount of alcohol or acetonitrile in the eluent used in the second stage. from separation.

Normally, the water: organic solvent ratio of the eluent in the first stage of separation is lower than the water: organic solvent ratio in the second stage of separation. Therefore, the eluent in the first separation step usually contains more organic solvent, preferably alcohol, more preferably methanol, than the eluent in the second separation step.

In embodiments in which the aqueous organic solvent used in each separation step has a different water ratio: organic solvent, the ratio of water: organic solvent of the eluent in the first stage of separation is usually from 0: 100 to 5:95. parts by volume, preferably from 0.1: 99.9 to 2.5: 97.5 parts by volume, more preferably from 0.25: 99.75 to 2:98 parts by volume, and most preferably from 0, 5: 99.5 to 1.5: 98.5 parts by volume. In these embodiments, the water: organic solvent ratio of the eluent in the second stage of separation is usually from 2:98 to 8:92 parts by volume, preferably from 3:97 to 7:93 parts by volume, more preferably from 4: 96 to 6:94 parts by volume, and even more preferably from 4.5: 95.5 to 5.5: 94.5 parts by volume.

In a particularly preferred embodiment where the aqueous organic solvent used in each separation step has a different content of organic solvent in water, the water: organic solvent ratio of the eluent in the first separation step is 0.5: 99.5 a 1.5: 98.5 parts by volume and the ratio of water: organic solvent of the eluent in the second stage of separation is from 4.5: 95: 5 to 5.5: 94.5 parts by volume.

It will be appreciated that the ratios between the water and the organic solvent in each separation step referred to above are average ratios within the entire chromatographic apparatus.

Normally, the ratio water: organic solvent of the eluent in each stage of separation is controlled by introducing water and / or organic solvent in one or more columns in the chromatographic apparatus used in the separation stages. Thus, for example, in order to achieve a lower water / organic solvent ratio in the first separation stage than in the second separation stage, water is usually introduced more slowly into the chromatographic apparatus used in the first stage of separation than in the second stage of separation. The second stage of separation.

In some embodiments, the essentially pure organic solvent and essentially pure water can be introduced at different points of the chromatographic apparatus used in each separation step. The relative flow rates of these two currents will determine the overall profile of the solvent in the chromatographic apparatus. In other embodiments, different organic solvent / water mixtures can be introduced at different points in each chromatographic apparatus used in each separation step. That will involve the introduction of two or more different organic solvent / water mixtures into the chromatographic apparatus used in a particular separation step, each organic solvent / water mixture having a different ratio of organic solvent: water. The relative flow rates and relative concentrations of the organic solvent / water mixtures in this embodiment will determine the overall profile of the solvent in the chromatographic apparatus used in that separation step.

In the chromatographic separation process of the invention, either (1) the intermediate product containing the PUFA product together with more polar components is collected as the refining stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation stage; or (2) the intermediate product containing the PUFA product together with less polar components is collected as the extract stream in the first separation step, and the PUFA product is collected as the refining stream in the second separation step. .

Embodiment (1) is suitable for purifying EPA from a feed mix.

This particularly preferred embodiment (1) is illustrated in Figure 2. A feed mix F comprising the PUFA product (B) and more polar (C) and less polar components (A) is purified in the first separation step. In the first separation stage, the less polar components (A) are removed as extract stream E1. The PUFA product (B) and the more polar components (C) are collected as refining stream R1. The refining stream R1 is the intermediate product which is then purified in the second separation step. In the second separation stage, the more polar components (C) are removed as refining stream R2. The PUFA product (B) is collected as E2 extract stream.

This embodiment is illustrated in more detail in Figure 4. Figure 4 is identical to Figure 2, except that the introduction points of the organic solvent desorbent (D) and water (W) into each chromatographic apparatus are shown. The organic solvent desorbent (D) and water (W) together constitute the eluent. Phase (D) is usually essentially pure organic solvent but, in some embodiments, it may be a mixture of organic solvent / water comprising mainly organic solvent. Phase (W) is normally essentially pure water but, in some embodiments, it may be a mixture of organic solvent / water comprising mainly water, for example a mixture of 98% water / 2% methanol.

A further illustration of this embodiment is shown in Figure 6. In this case there is no independent water injection point, and an aqueous organic solvent desorbent is injected into (D) instead.

The stream separation of raffinate and extract can be facilitated by modifying the desorption power of the eluent within each chromatographic apparatus. This can be achieved by introducing the organic solvent component (or rich in organic solvent) of the eluent and the water (or water-rich) component at different points in each chromatographic apparatus. Therefore, normally, the organic solvent is introduced upstream of the extract extraction point and the water is introduced between the extraction point of extract and the point of introduction of the feed in the chromatographic apparatus, with respect to the flow of eluent in the system. This is shown in Figure 4.

Typical solvents for use in this embodiment are most preferred aqueous alcohols or aqueous acetonitrile, preferably aqueous methanol.

Normally, in this embodiment, the aqueous organic solvent eluent used in the first stage of separation contains more organic solvent than the eluent used in the second stage of separation, i.e., the ratio of water: organic solvent in the first stage is lower that the water ratio: organic solvent in the second stage.

In this embodiment, the first refining stream in the first separation stage is normally removed downstream of the introduction point of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the flow of eluent.

In this embodiment, the first extract stream in the first separation step is usually removed upstream of the introduction point of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the eluent flow.

In this embodiment, the second refining stream in the second separation step is usually removed downstream of the introduction point of the intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.

In this embodiment, the second extract stream in the second separation step is normally collected upstream of the introduction point of the intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.

Normally in this embodiment, the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the first separation stage upstream of the point of elimination of the first extract stream, with respect to the eluent flow.

Normally in this embodiment, when water is introduced into the chromatographic apparatus used in the first separation stage, the water is introduced into the chromatographic apparatus used in the first separation stage upstream of the introduction point of the feed mixture but downstream of the elimination point of the first extract stream, with respect to the eluent flow.

Normally in this embodiment, the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the second stage of separation upstream of the point of elimination of the second extract stream, with respect to the flow of eluent.

Normally in this embodiment, when water is introduced into the chromatographic apparatus used in the second stage of separation, the water is introduced into the chromatographic apparatus used in the second stage of separation. upstream of the introduction point of the intermediate product but downstream of the elimination point of the second extract stream, with respect to the eluent flow.

The preferred embodiment (2) is suitable for purifying DHA from a feed mix.

Embodiment (2) is illustrated in Figure 3. A feed mix F comprising the PUFA product (B) and more polar (C) and less polar (A) components is purified in the first separation step. In the first separation stage, the more polar components (C) are removed as refining stream R1. The PUFA product (B) and the less polar components (A) are collected as E1 extract stream. The extract stream E1 is the intermediate product that is then purified in the second separation step. In the second separation stage, the less polar components (A) are removed as stream of E2 extract. The PUF product ^a (B) is collected as refining stream R2.

This embodiment is illustrated in more detail in Figure 5. Figure 5 is identical to Figure 3, except that the introduction points of the organic solvent desorbent (D) and water (W) are shown in each chromatographic apparatus. As above, phase (D) is usually essentially pure organic solvent, but, in some embodiments, it may be a mixture of organic solvent / water comprising mainly organic solvent. The phase (W) is normally essentially pure water but, in some embodiments, it can be a mixture of organic solvent / water comprising mainly water, for example a mixture of 98% water / 2% methanol.

A further illustration of this embodiment is shown in Figure 7. In this case there is no independent water injection point, and an aqueous organic solvent desorbent is injected into (D) instead.

Typical solvents for use in this embodiment are most preferred aqueous alcohols or aqueous acetonitrile, preferably aqueous methanol.

Normally in this embodiment, the aqueous organic solvent eluent used in the first separation step contains less organic solvent than the eluent used in the second separation stage, ie, the water: organic solvent ratio in the first stage of separation is greater than in the second stage of separation. In this embodiment the first refining stream in the first separation step is usually removed downstream of the introduction point of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the flow of eluent.

In this embodiment, the first extract stream in the first separation step is usually removed upstream of the introduction point of the feed mixture into the chromatographic apparatus used in the first separation step, with respect to the eluent flow.

In this embodiment, the second refining stream in the second separation step is usually removed downstream of the introduction point of the intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.

In this embodiment, the second extract stream in the second separation step is normally collected upstream of the introduction point of the intermediate product in the chromatographic apparatus used in the second separation step, with respect to the eluent flow.

Normally in this embodiment, the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the first separation stage upstream of the point of elimination of the first extract stream, with respect to the eluent flow.

Normally in this embodiment, when water is introduced into the chromatographic apparatus used in the first separation stage, the water is introduced into the chromatographic apparatus used in the first separation stage upstream of the introduction point of the feed mixture but downstream of the elimination point of the first extract stream, with respect to the eluent flow.

Normally in this embodiment, the organic solvent or aqueous organic solvent is introduced into the chromatographic apparatus used in the second stage of separation upstream of the point of elimination of the second extract stream, with respect to the flow of eluent.

Normally in this embodiment, when water is introduced into the chromatographic apparatus used in the second stage of separation, the water is introduced into the chromatographic apparatus used in the second stage of separation upstream of the intermediate product introduction point but downstream of the point of elimination of the second extract stream, with respect to the eluent flow.

In a preferred embodiment of the invention, each of the simulated or actual movable bed chromatography apparatus used in the first and second separation stages consists of eight chromatographic columns. These are called columns 1 to 8. In each apparatus the eight columns are arranged in series so that the lower part of the column 1 is attached to the upper part of the column 2, the lower part of the column 2 is attached to the top of column 3 ... etc ... and the bottom of column 8 is attached to the top of column 1. These unions can optionally be by a retention vessel, with a recirculation stream at inside of the next column. The flow of eluent through the system is from column 1 to column 2 to column 3 etc. The effective flow of adsorbent through the system is from column 8 to column 7 to column 6 etc.

A most preferred embodiment is illustrated in Figure 8. A feed mix F comprising the PUFA product (B) and more polar (C) and less polar components (A) is introduced into the top of column 5 in the chromatographic apparatus used in the first separation stage. The organic solvent desorbent is introduced into the top of column 1 of the chromatographic apparatus used in the first separation step. Water is introduced into the top of column 4 of the chromatographic apparatus used in the first separation step. In the first separation stage, the less polar components (A) are removed as extract stream E1 from the bottom of column 2. The PUFA product (B) and the more polar components (C) are removed as current of refining R1 from the lower part of column 7. The refining stream R1 is the intermediate product which is then purified in the second separation stage, when it is introduced into the chromatographic apparatus used in the second stage of separation in the top of column 5. The organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the second separation stage. Water is introduced into the upper part of column 4 in the chromatographic apparatus used in the second separation stage. In the second separation stage, the more polar components (C) are removed as refining stream R2 at the bottom of column 7. The PUFA product (B) is collected as stream of E2 extract at the bottom of the column. column 2.

In this most preferred embodiment, the organic solvent is usually introduced into the top of column 1 of the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, water is usually introduced into the top of column 4 of the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, the organic solvent is usually introduced into the top of column 1 of the chromatographic apparatus used in the second separation step.

In this most preferred embodiment, the organic solvent is usually introduced into the top of column 4 of the chromatographic apparatus used in the second separation step.

In this most preferred embodiment, the feed stream is usually introduced into the top of column 5 of the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, a first raffinate stream is normally collected as the intermediate product from the bottom of column 7 of the chromatographic apparatus used in the first separation step. This intermediate product is then purified in the second separation step and is usually introduced in the upper part of column 5 of the chromatographic apparatus used in the second separation step. The first refining stream can optionally be collected in a container before being purified in the second separation step.

In this most preferred embodiment, a first extract stream is usually removed from the bottom of column 2 of the chromatographic apparatus used in the first separation step. The first extract stream can optionally be collected in a container and reintroduced to the top of column 3 of the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, a second raffinate stream is usually removed from the bottom of column 7 of the chromatographic apparatus used in the second separation step.

In this most preferred embodiment, a second extract stream is normally collected from the bottom of column 2 of the chromatographic apparatus used in the second separation step. This second extract stream usually contains the purified PUFA product. The second extract stream can optionally be collected in a container and reintroduced to the top of column 3 of the chromatographic apparatus used in the second separation step.

In this most preferred embodiment, the eluent used is usually aqueous alcohol, preferably aqueous methanol. The water: alcohol ratio is usually from 0.5: 99.5 to 6:94 parts by volume.

Normally, in this most preferred embodiment, the ratio of water: organic solvent in the chromatographic apparatus used in the first stage of separation is lower than the ratio of water: organic solvent in the chromatographic apparatus used in the second stage of separation. Therefore, the eluent in the first separation step usually contains more organic solvent than the eluent used in the second separation stage.

The ratio of water: organic solvent in the first stage of separation is usually from 0.5: 99.5 to 1.5: 98.5 parts by volume. The ratio of water: organic solvent in the second stage of separation is usually from 2:98 to 6:94 parts by volume.

Although the embodiment of Figure 8 is configured as shown in Figure 10a, the configuration shown in Figures 10b and 10c could also be used in this embodiment.

A further preferred embodiment is illustrated in Figure 9. A feed mix F comprising the PUFA product (B) and more polar (C) and less polar components (A) is introduced into the top of the column. in the chromatographic apparatus used in the first stage of separation. The aqueous organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the first separation step. In the first separation stage, the less polar components (A) are removed as extract stream E1 from the bottom of column 2. The PUFA product (B) and the more polar components (C) are removed as current of refining R1 from the bottom of column 7. The refining stream R1 is the intermediate product that is purified in the second stage of separation when it is introduced in the upper part of column 4 of the chromatographic apparatus used in the second stage from separation. The aqueous organic solvent desorbent is introduced into the top of column 1 in the chromatographic apparatus used in the second stage of separation. In the second separation stage, the more polar components (C) are removed as refining stream R2 at the bottom of column 7. The PUFA product (B) is collected as stream of E2 extract at the bottom of the column. column 2.

In this most preferred embodiment, the aqueous organic solvent is usually introduced into the top of column 1 in the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, the aqueous organic solvent is usually introduced into the top of column 9 in the chromatographic apparatus used in the second separation step.

In this most preferred embodiment, the feed stream is usually introduced into the top of column 5 in the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, a first raffinate stream is normally collected as the intermediate product from the bottom of column 7 of the chromatographic apparatus used in the first separation step. This intermediate product is then purified in the second separation step and is usually introduced in the upper part of column 5 of the chromatographic apparatus used in the second separation step. The first refining stream can optionally be collected in a container before being purified in the second separation step.

In this most preferred embodiment, a first extract stream is usually removed from the bottom of column 2 of the chromatographic apparatus used in the first separation step. The first extract stream can optionally be collected in a container and a part reintroduced into the top of column 3 of the chromatographic apparatus used in the first separation step. The flow rate of liquid recirculation collected by the extract stream in the first stage of separation back into the chromatographic apparatus used in the first stage of separation is the rate at which the liquid is pumped from this container into the upper part. from column 3.

In this most preferred embodiment, a second raffinate stream is usually removed from the bottom of column 7 of the chromatographic apparatus used in the first separation step.

In this most preferred embodiment, a second extract stream is usually collected from the bottom of column 2 of the chromatographic apparatus used in the first separation step. This second extract stream usually contains the purified PUFA product. The second extract stream can optionally be collected in a container and a part reintroduced into the top of column 3 of the chromatographic apparatus used in the first separation step. The flow rate of liquid recirculation collected by the extract stream from the second stage of separation back into the chromatographic apparatus used in the second stage of separation is the rate at which the liquid is pumped from this container into the interior of the part. top of column 3.

In this most preferred embodiment, the eluent used is usually aqueous alcohol, preferably aqueous methanol. The water: alcohol ratio is usually from 0.5: 99.5 to 6:94 parts by volume.

Normally, in this most preferred embodiment, the ratio of water: organic solvent in the chromatographic apparatus used in the first stage of separation is less than the ratio of water: organic solvent in the chromatographic apparatus used in the second stage of separation. Therefore, the eluent used in the first separation step usually contains more organic solvent than the eluent used in the second separation stage.

The ratio of water: organic solvent in the first stage of separation is usually from 0.5: 99.5 to 1.5: 98.5 parts by volume. The ratio of water: organic solvent in the second stage of separation is usually from 2:98 to 6:94 parts by volume.

Although the embodiment of Figure 9 is configured as shown in Figure 10a, the configuration shown in Figures 10b and 10c could also be used in this embodiment.

The process of the invention allows much higher PUFA product purities to be achieved than has been possible with conventional chromatographic techniques. The PUFA products produced by the process of the invention also have particularly advantageous impurity profiles, which are quite different from those observed in oils prepared by known techniques. Therefore, compositions comprising a PUFA product, for example one obtainable by the process of the present invention, are also disclosed herein.

In practice, the process of the present invention will generally be controlled by a computer. The present invention, therefore, also provides a computer program for controlling a chromatographic apparatus as defined herein, the computer program containing a code means which, when executed, commands the apparatus to carry out the process of the invention.

The following examples illustrate the invention.

Examples

Example 1

A feed charge derived from fish oil (55% by weight of EPA, 5% by weight of DHA EE) is fractionated using a real cell-bed chromatography system using bound C18 silica gel (particle size). pm) as stationary phase and aqueous methanol as eluent according to the system illustrated schematically in Figure 9. 8 columns are connected in series (diameter: 4.6 mm, length 250 mm), as shown in Figure 9 .

The feed mixture was passed through the SMB apparatus in a first stage of separation. In the first stage of separation, the process conditions were adjusted to purify the EPA from DHA and other slow-moving impurities. The EPA, together with other fast-moving impurities, was collected in the refining stream as an intermediate product. The extract stream containing DHA and other slow-moving impurities was rejected. The process conditions of the SMB device were then adjusted for the second stage of separation. In the second stage of separation, the process conditions were adjusted to purify the EPA from the faster advancing impurities. The aqueous methanol eluent used in the second separation step had a higher ratio of water: organic solvent than the aqueous methanol eluent used in the first separation stage. The intermediate product was introduced into the SMB apparatus as the feed mixture in the second stage of separation. High purity EPA was collected as the extract stream. The refining stream containing fast-moving impurities was rejected.

The EPA was produced with a final purity of ~ 95% and a recovery of ~ 95%.

An HPLC analysis of the raw material is shown in Figure 11.

The HPLC analyzes of the refining (R) and extract (E) streams of the first separation stage are shown in Figure 12.

The HPLC analyzes of the refining (R) and extract (E) streams from the second separation stage are shown in Figure 13. A more detailed HPLC analysis of the extract stream from the second separation stage is shown in Figure 14

Figures 12 and 13 also show the process conditions for the first and second separation steps.

Reference example 1

An experiment was conducted to compare the amount of environmental contaminants present in two PUFA products produced by SMB with similar oils prepared by distillation. The profiles of the contaminants of the oils are shown in the following Table 1.

Table 1

Reference example 2

An experiment was carried out to determine the amount of isomeric impurities present in an oil prepared by SMB in comparison with an equivalent oil prepared by distillation.

A GC trace of the DHA-rich oil prepared by SMB is shown in Figure 15. There is no evidence of isomeric impurities in the GC footprint.

A GC trace of the oil prepared by distillation is shown in Figure 16. The four peaks with elution times longer than the DHA peak correspond to DHA isomers. From the GC trace it can be seen that the oil prepared by distillation contains approximately 1.5% by weight of isomeric impurities.

Reference example 3

Two products rich in EPA produced by SMB were compared with oils rich in EPA produced by distillation. The analysis of the weight% of its PUFA components are shown below.

Reference example 4

A product rich in EPA / DHA produced by SMB was compared with an oil rich in EPA / DHA produced by distillation. The analysis of the percentage by weight of its PUFA components is shown below.

Claims (13)

A chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product from a feed mixture, a process comprising the steps of: (i) purifying the feed mixture in a first step of separation in a simulated or real movable bed chromatography apparatus having a plurality of bound chromatography columns containing, as eluent, an aqueous organic solvent, to obtain an intermediate product ; Y (ii) purifying the intermediate product obtained in (i) in a second separation step using a simulated or actual movable bed chromatography apparatus having a plurality of bound chromatography columns containing, as eluent, an aqueous organic solvent, for obtain the PUFA product; in which (a) The first and second separation steps are carried out sequentially in the same chromatography apparatus, the intermediate product being recovered between the first and second separation steps and the process conditions being adjusted in the chromatography apparatus between the first and second steps. separation steps so that the PUFA product is separated from different components of the feed mixture in each separation step; or (b) the first and second separation steps are carried out in a first and second independent chromatography apparatus respectively, introducing the intermediate product obtained from the first separation step in the second chromatography apparatus, and separating the PUFA product from different components of the feed mixture in each stage of separation; Y wherein the aqueous organic solvent eluent used in each separation step has a volume ratio of water: different organic solvent; Y wherein (1) the intermediate product is collected as the refining stream in the first separation step, and the PUFA product is collected as the extract stream in the second separation step; or (2) the intermediate product is collected as the extract stream in the first separation step, and the PUFA product is collected as the refining stream in the second separation step. 2. A process according to claim 1, wherein each apparatus has an extract stream and a raffinate stream from which liquid can be collected from said plurality of attached chromatography columns. 3. A process according to claim 1 or 2, wherein the intermediate product recovered from the first separation stage is enriched in the PUFA product as compared to the feed mixture. 4. A process according to any one of the preceding claims, wherein the PUFA product is separated from less polar components of the feed mixture in the first separation stage, and the PUFA product is separated from more polar components. of the feed mixture in the second stage of separation. 5. A process according to any one of the preceding claims, wherein the PUFA product comprises at least one PUFA w-3 and wherein the PUFA product preferably comprises EPA and / or DHA. A process according to any one of the preceding claims, wherein the eluent is a mixture of water and an alcohol, an ether, an ester, a ketone or a nitrile, and wherein the eluent is preferably a mixture of water and methanol. 7. A process according to any one of the preceding claims, wherein the feed mixture is a fish oil or a feedstock derived from fish oil, the PUFA product is EPA or EPA ethyl ester, and the PUFA product is produced with a purity greater than 90%, preferably with a purity greater than 95% and more preferably with a purity greater than 97%. 8. A process according to any one of the preceding claims, wherein (a) part of the extract stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation stage; I (b) part of the refining stream from the apparatus used in the first separation step is recirculated back into the interior of the apparatus used in the first separation stage; I (c) part of the extract stream from the apparatus used in the second separation step is recirculated back into the interior of the apparatus used in the second separation stage; I (d) part of the raffinate stream from the apparatus used in the second stage of separation is recirculated back into the interior of the apparatus used in the second stage of separation. 9. A process according to any one of the preceding claims, wherein the ratio of water: organic solvent used in each stage of separation is adjusted such that the PUFA product can be separated from different components of the mixture of feeding in each stage of separation. 10. A process according to any one of the preceding claims, wherein the ratio in volume of water: organic solvent of the eluent in the first stage of separation is less than the ratio by volume of water: organic solvent of the eluent in the Second stage of separation. 11. A process according to any one of the preceding claims, wherein the volume ratio of water: organic solvent of the eluent in the first separation step is from 0.5: 99.5 to 1.5: 98, 5 parts by volume, and the volume ratio of water: organic solvent of the eluent in the second stage of separation is from 4.5: 95: 5 to 5.5: 94.5 parts by volume. 12. A process according to any one of the preceding claims, wherein the ratio in volume of water: organic solvent of the eluent in the first and second stages of separation is controlled by introducing water and / or organic solvent in one or more columns in the apparatuses used in the first and second stages of separation. 13. A computer program for controlling a chromatography apparatus as defined in any one of claims 1 to 12, computer program containing a code medium which, when executed, instructs the apparatus to carry out a process in accordance with any one of claims 1 to 12.